Our research is focused on the hypothesis that the ancient and divergent classes of plant actin genes have been preserved throughout vascular plant evolution because they have unique patterns of gene regulation and/or encode proteins with unique functions. During the last grant period we isolated, sequenced, and characterized the ten members of the Arabidopsis actin gene family (ACT1, 2, 3, 4, 5, 7, 8, 9psi, 11, & 12). They fall into six subclasses which were approximately as divergent as vertebrate muscle actin is from cytoplasmic actin, but the plant proteins had much higher levels of charged residue variability. Based on gene specific Northerns and RT-PCR analysis of RNA expression and 5' region/reporter fusions examined in hundreds of independent transgenic plants, eight of the genes in five subclasses were expressed at very high levels in at least one cell type. These five subclasses had very distinct and different expression patterns covering all the tissues and developmental stages of Arabidopsis (e.g. the ACT2/ACT8 subclass was constitutive in vegetative tissues, while the ACT1/ACT3 subclass was expressed in organ primordia and mature pollen). An initial characterization of Arabidopsis profilin sequences demonstrated the presence of a large and complex gene family which included constitutive (PRF1, 2, 3) and pollen specific members (PRF4). In light of these and other data on the diverse plant actins and profilins we propose a second hypothesis for our future work: there are concordantly expressed subclasses of actins and actin binding proteins (e.g. profilins), which have evolved distinct protein-protein interactions. Based on these two hypotheses our specific aims for the next grant period are: (l) to demonstrate that the various actin genes or subfamilies are required for normal Arabidopsis growth and development by characterizing the phenotype of plants deficient in actin expression (from a T-DNA insertion library and by antisense suppression and co-suppression); (2) To determine if the major cis-elements controlling tissue specific expression are in the promoter region, mRNA leader or leader intron; (3) to determine the detailed expression patterns of a few representative members of the diverse profilin subclasses (i.e. PRF1 and PRF4); (4) to initiate experiments which will connect the origin of the diverse actin lineages with macroevolutionary events (e.g. ACT1 with the origin of meristems); and (5) to compare the strength of protein-protein interaction between actin-profilin pairs that are co-expressed, with those that are not. This work will expand our basic understanding of the eukaryotic cytoskeleton and enhance our ability to manipulate the properties of higher plants.